The long term objective of this work is to gain deeper insights into the mechanisms of transcriptional activation by nuclear hormone receptors.The specific goal of this project is to identify and study the target factors that specifically interact with and mediate the transactivation function of nuclear receptors in vivo (referred as nuclear receptor interacting factors, NRIFs). To achieve this goal, major efforts will be focused on further characterization of four putative NRIF cDNA clones isolated from a yeast two-hybrid screening, which encode proteins specifically interacting with one or several of the nuclear receptors in a ligand-dependent manner. Immunocytochemical analysis will be performed to examine whether these NRIFs are nuclear proteins. These candidate NRIFs will also he studied for their interactions with nuclear receptors both in vitro (using binding and gel shift assays) and in vivo (using co-immunoprecipitation and transfection assays) to examine their possible role(s) in receptor actions. In another effort, a genetic approach will be used to identify factors that interact with the N-terminal A/B regions of thyroid hormone receptor beta2(TR beta2) and retinoic acid receptor alpha1 (RARalpha1) and act to mediate the transcriptional activation function of these two A/B regions in vivo (referred as A- region interacting factors, ABIFs). The identification and study of the NRIFs and ABIFs should not only facilitate further understanding of receptor functions and hormone actions, but also may have broad relevance to regulation of transcription in general.